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Y. Garfias, M. Vazquez-Membrillo, A. Rojas-Diaz, X. Mira, A. Robles-Contreras, E. Lopez-Star, M. C. Jimenez-Martinez, V. M. Bautista; Doxycycline Enhances Apoptosis and Increases TNF-Alpha Expression in Pterygial Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2400.
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© ARVO (1962-2015); The Authors (2016-present)
In a previous study we have shown that the tetracycline doxycycline had an anti-apoptotic effect on primary culture of healthy conjunctival cells (ARVO 2009), etc). The objective of the present study is to determine the effect of doxycycline in the apoptosis and TNF-alpha expression in pterygial fibroblasts (PF)
The fibroblasts were obtained from primary pterygia. All the experiments were performed at 3-5 passages, when the cells reached at least 80% of confluence. The phenotypical characterization of the cells was performed by flow cytometry (FC). The PF were incubated with different doses of doxycycline (100, 200, 400 mM); after incubation with doxycycline, the cells were harvested and FC was performed to identify apoptosis and apoptosis related proteins. TNF-alpha was analyzed by cytometric bead arrays technique.
Doxycycline increased PF apoptosis (35.5±10%) compared to non stimulated PF (5.9±2% ), in a dose dependent manner. The protein expression related with apoptosis showed an increase compared to controls, as follows: Bax 17% in controls vs. 62% in doxycycline stimulated PF; Bcl-2, 99% in controls vs. 81% in doxycycline stimulated PF, and Bcl-X 4% in controls vs. 26% in doxycycline stimulated PF. The ratios between apoptotic/non-apoptotic molecules were as follows: for Bax/Bcl-2, 0.17 in controls vs. 0.79 in doxycycline stimulated PF; Bax/Bcl-X, 1.13 in controls vs. 2.35. When TNF-alpha was analyzed, doxycycline stimulated PF secreted 16.2 pg/ml vs. controls which secreted 3.3 pg/ml of this cytokine.
Taken together all these results suggest that doxycycline has a positive effect on PF apoptosis increasing Bax/Bcl2 and Bax/Bcl-X indexes, which were dose dependent. By the other hand, this drug induces the secretion of TNF-alpha which is a pro-inflammatory cytokine involved in cell death, the increase of this molecule could partially explain the apoptotic phenomenon induced by doxycycline. In conclusion, this tetracycline would be an alternative therapy in the treatment for primary and recurrent pterygium.
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